Minimally invasive localization of oncolytic herpes simplex viral therapy of metastatic pleural cancer

Herpes simplex virus-1 (HSV-1) oncolytic therapy and gene therapy are promising treatment modalities against cancer. NV1066, one such HSV-1 virus, carries a marker gene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine whether NV1066 is cytotoxic to lung cancer and whether EGFP is a detectable marker of viral infection in vitro and in vivo. We further investigated whether EGFP expression in infected cells can be used to localize the virus and to identify small metastatic tumor foci (<1 mm) in vivo by means of minimally invasive endoscopic systems equipped with fluorescent filters. In A549 human lung cancer cells, in vitro viral replication was determined by plaque assay, cell kill by LDH release assay, and EGFP expression by flow cytometry. In vivo, A549 cells were injected into the pleural cavity of athymic mice. Mice were treated with intrapleural injection of NV1066 or saline and examined for EGFP expression in tumor deposits using a stereomicroscope or a fluorescent thoracoscopic system. NV1066 replicated in, expressed EGFP in infected cells and killed tumor cells in vitro. In vivo, treatment with intrapleural NV1066 decreased pleural disease burden, as measured by chest wall nodule counts and organ weights. EGFP was easily visualized in tumor deposits, including microscopic foci, by fluorescent thoracoscopy. NV1066 has significant oncolytic activity against a human NSCLC cell line and is effective in limiting the progression of metastatic disease in an in vivo orthotopic model. By incorporating fluorescent filters into endoscopic systems, a minimally invasive means for diagnosing small metastatic pleural deposits and localization of viral therapy for thoracic malignancies may be developed using the EGFP marker gene inserted in oncolytic herpes simplex viruses.     

Cisplatin-induced GADD34 upregulation potentiates oncolytic viral therapy in the treatment of malignant pleural mesothelioma

BACKGROUND: NV1066, a replication-competent oncolytic herpes simplex virus type 1 (HSV-1) attenuated by a deletion in the gene gamma(1)34.5, preferentially replicates in and kills malignant cells. gamma(1)34.5 encodes ICP34.5, a viral protein essential for productive replication, which has homology with mammalian stress response induced GADD34 (growth arrest and DNA damage-inducible protein). We hypothesized that cisplatin upregulates GADD34 expression, which enhances NV1066 replication and oncolysis. METHODS: Ten human malignant pleural mesothelioma (MPM) cell lines were infected with NV1066 at multiplicities of infection (MOI; ratio of viral particles per tumor cell) 0.005 to 0.8 in vitro, with and without cisplatin (1 to 4 microM). In the MPM cell line VAMT, viral replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 induction by quantitative RT-PCR and Western blot. Synergistic efficacy was confirmed by the isobologram and combination index methods of Chou-Talalay. GADD34 upregulation by cisplatin was inhibited with GADD34 siRNA to further confirm the synergistic efficacy dependence with GADD34. RESULTS: Combination therapy with NV1066 and cisplatin showed strong synergism in epithelioid (H-2452, H-Meso), sarcomatoid (H-2373, H-28), and biphasic (JMN, Meso-9, MSTO-211H) MPM cell lines, and an additive effect in others. In VAMT cells combination therapy enhanced viral replication 4 to 11-fold (p < 0.01) and cell kill 2 to 3-fold (p < 0.01). Significant dose reductions for both agents (2 to 600-fold) were achieved over a wide range of therapeutic-effect levels (LD50-LD99) without compromising cell kill. Synergistic cytotoxicity correlated with GADD34 upregulation (2 to 4-fold, p < 0.01) and was eliminated following transfection with GADD34 siRNA. CONCLUSION: Cisplatin-induced GADD34 expression selectively enhanced the cytotoxicity of the gamma(1)34.5-deficient oncolytic virus, NV1066. This provides a cellular basis for combination therapy with cisplatin and NV1066 to treat MPM and achieve synergistic efficacy, while minimizing dosage and toxicity.     

Estrogen enhances the efficacy of an oncolytic HSV-1 mutant in the treatment of estrogen receptor-positive breast cancer

Oncolytic herpes simplex virus-1 (HSV-1) mutants selectively replicate in and lyse tumor cells. Viral replication is dependent on the cellular proliferative mechanism. Estrogen increases cellular proliferation and decreases apoptosis in estrogen receptor-positive (ER+) human breast cancer cells. We hypothesize that the cellular changes produced by estrogen may enhance oncolytic viral replication and improve the treatment of ER+ breast cancer cells. Estrogen increased proliferation and replication of the HSV-1 mutant, NV1066, in ER+ breast cancer cells. Additionally, cells grown with estrogen had lower rates of apoptosis and higher bcl-2 levels at baseline and after infection. Estrogen enhanced the oncolytic effect of NV1066, with cell kills of 95% and 97% at MOIs of 0.1 and 0.5, compared to 53 and 87% respectively without estrogen (p<0.001). Therapy of ER+ human breast cancer cells with a replication-competent HSV-1 mutant is improved in the presence of estrogen, in contrast to more standard therapies, such as chemotherapy and radiation, which demonstrate decreased efficacy in similar conditions. These data provide the mechanistic basis for the use of oncolytic HSV-1 in patients with hormone receptor-positive breast cancer, particularly if the disease progresses with conventional therapies.     

Effect of a caspase inhibitor, zVADfmk, on the inhibition of breast cancer cells by herpes simplex virus type 1

The oncolytic effects of herpes simplex virus-1 (HSV-1) are limited, possibly because of premature death of infected cells by apoptosis, which limits the amount of progeny virus that is produced. It has been proposed that inhibition of apoptosis in infected tumor cells would allow increased viral persistence, replication and therapeutic effect. To test this hypothesis, we infected monocyte chemoattractant factor-7 (MCF-7) and MDA-MB-231 breast cancer cells with HSV-1 strain 17(+) and 17Δγ34.5 in the presence or absence of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk), a pan-caspase inhibitor. At low doses of HSV-1 strain 17(+) and 17Δγ34.5, the growth of MCF-7 cells was reduced to 37% or 42%, respectively, of uninfected cells. However, when cells were infected in the presence of zVADfmk, cell growth was further reduced to 24 and 33%. Similar results were seen in MDA-MB-231 cells. Cells treated with zVADfmk contained roughly 10 times more infectious viral particles than cells infected without zVADfmk, as shown by both plaque-forming and quantitative polymerase chain reaction assays. To model the situation within an infected tumor, supernatant fluids were collected from infected and non-infected cell cultures and then passed to non-infected cells. In the presence of zVADfmk, the cell growth inhibitory effect became stronger with repeated passages and was attributed to viral replication, because it could be prevented by anti-HSV antibody. These results suggest that caspases represent a novel target for drugs that increase the therapeutic efficacy of oncolytic herpes viruses against breast cancer.     

Upregulation of thioredoxin reductase 1 in human oral squamous cell carcinoma

Lymphoma is not a common focus for viral therapy as many researchers do not consider lymphoma as a suitable target for viral vectors. In the present study, the infection, replication and cytotoxicity of herpes simplex virus (HSV) and adenovirus vectors were screened and evaluated in different lymphoma cell lines. Three recombinant viruses, BAC-HSV-1-eGFP, Adv-eGFP and an NV1020-like oncolytic HSV-1 virus strain named BAC-HSV-1-eGFP-delIRs, inserted with green fluorescent protein (GFP) as a reporter, were applied to evaluate the efficiency of viral infection and replication and cytotoxicity to lymphoma cells. Four types of lymphoma cell lines (SNK-6, Jurkat, Raji and Hut 78) were examined. We found that the HSV-1 vector, BAC-HSV-1-eGFP, was able to infect and replicate in the three lymphoma cell lines (Jurkat, Raji and Hut 78) and inhibit the growth of these cells more efficiently than adenovirus vector Adv-eGFP. The sensitivity of the four types of lymphoma cell lines to the viral vectors was different. The human cutaneous TL cell line Hut 78 was more sensitive to the viral vectors than the other cell lines. However, the human NK/T lymphoma cell line SNK-6 was not infected by any of the viral vectors and did not allow replication of the viruses. In conclusion, lymphoma may be a potential target for HSV-1 vector-mediated viral therapy.     

Use of carrier cells to deliver a replication-selective herpes simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) remains localized within the peritoneal cavity in a large number of patients, lending itself to i.p. approaches of therapy. In the present study, we investigated the effect of replication-selective herpes simplex virus-1 (HSV-1) used as an oncolytic agent against EOC and the use of human teratocarcinoma PA-1 as carrier cells for i.p. therapy. HSV-1716, a replication-competent attenuated strain lacking ICP34.5, caused a direct dose-dependent oncolytic effect on EOC cells in vitro. A single i.p. administration of 5 x 10(6) plaque-forming units resulted in a significant reduction of tumor volume and tumor spread and an increase in survival in a mouse xenograft model. PA-1 cells supported HSV replication in vitro and bound preferentially to human ovarian carcinoma surfaces compared with mesothelial surfaces in vitro and in vivo. In comparison with the administration of HSV-1716 alone, irradiated PA-1 cells, infected at two multiplicities of infection with HSV-1716 and injected i.p. at 5 x 10(6) cells/animal, led to a significant tumor reduction in the two models tested and the significant prolongation of mean survival in one model. Histological evaluation revealed extensive necrosis in tumor areas infected by HSV-1716. Immunohistochemistry against HSV-1 revealed areas of viral infection within tumor nodules, which persisted for several weeks after treatment. Administration of HSV-infected PA-1 carrier cells resulted in larger areas of tumor infected by the virus. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on EOC, which may be further enhanced by the utilization of a delivery system with carrier cells, based on amplification of the viral load and possibly on preferential binding of carrier cells to tumor surfaces.     

Cytokine-secreting herpes viral mutants effectively treat tumor in a murine metastatic colorectal liver model by oncolytic and T-cell-dependent mechanisms

In this model of hepatic micrometastases, the antitumor efficacy and role of the T-cell and natural killer (NK) cell populations were studied for oncolytic herpes simplex virus type-1 (HSV-1) viral mutants containing the granulocyte-monocyte colony stimulating factor (GM-CSF (NV1034)) or interluken-12 (IL-12 (NV1042)) cytokine genes. These were compared to saline and control virus (NV1023) in vitro and in vivo. HSV-1 mutants were assessed for cytotoxicity, replication and cytokine expression in CT-26 cells. A syngeneic micrometastatic liver model was then established in naive and immune cell-depleted animals to assess the antitumor efficacy of these viruses. In vitro cytotoxicity and viral replication were similar for each virus, resulting in greater than 80 and 98% cytotoxicity at multiplicity of infection of 1 and 10, respectively. Peak viral titers were 25- to 50-fold higher than initial titer and were not significantly different between viruses. In vivo, all three viruses reduced metastases relative to control, but cytokine-secreting viruses did so with greater efficacy compared to NV1023. This effect was abrogated by T-cell depletion, but not NK-cell depletion. Single-agent therapy with oncolytic viral agents containing GM-CSF or IL-12 is effective in a murine model of liver metastases and likely involves direct viral oncolysis and actions of specific immune effector cells.     

Deficiency of caspase 3 in tumor xenograft impairs therapeutic effect of measles virus Edmoston strain

The oncolytic measles virus Edmonston (MV-Edm) strain shows considerable oncolytic activity against a variety of human tumors. In this study, we report MV-Edm is able to trigger apoptosis pathways in infected tumor cells and elucidate the roles of cellular apoptosis in the whole oncolytic process. We also show that activated caspase 3, a key executioner of apoptosis, plays key roles in the oncolytic virotherapy. Activated caspase 3 can accelerate viral replication in cervical cancer cells and enhance the killing effects of the virus. Deficiency of caspase 3 either in tumor cells or in tumor xenograft significantly desensitized tumor to oncolysis with MV-Edm. In the infected cells, caspase 3 regulates interferon α release, which can inhibit viral replication in neighboring tumor cells. We propose that caspase-3 activation enhances the oncolytic effects of MV-Edm, thus inhibiting tumor growth in mice.     

Systemic therapy of spontaneous prostate cancer in transgenic mice with oncolytic herpes simplex viruses

Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12-expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies.     

Potent systemic antitumor activity from an oncolytic herpes simplex virus of syncytial phenotype

Conditionally replicating (oncolytic) viruses, which selectively replicate in tumor cells but not in normal cells, show great promise as antitumor agents for cancer therapy. The principal antitumor activity of these viruses derives from their replication within tumor cells, which results in cell destruction and the production of progeny virions that can spread to adjacent tumor cells. However, one potential limitation of this approach is that viral gene deletions conferring tumor selectivity also result frequently in reduced potency of the virus in tumors. Therefore, strategies designed to enhance the potency of current oncolytic viruses will likely increase their chance of clinical success. Here we report the construction of an oncolytic herpes simplex virus (HSV) of which the infection also causes strong cell membrane fusion (syncytial formation). In vitro characterization on a variety of human tumor cells of different tissue origins showed that the plaques from this virus (Fu-10) are phenotypically unique and are significantly larger than those from the parental G207 virus, a well-characterized oncolytic HSV lacking fusogenic function. Furthermore, the syncytial formation caused by this virus depended on HSV replication, indicating that cell membrane fusion will only occur in dividing cells (such as tumor cells) where the virus can undergo a full infection cycle but not in normal cells where the viral replication is restricted. Systemic administration of Fu-10 into mice with established lung metastatic breast cancer resulted in a dramatic therapeutic effect. These studies demonstrate that incorporation of fusogenic function into an oncolytic virus can significantly increase the potency of viral oncolysis; this may lead to an enhanced clinical performance, especially in late-stage cancer patients.     

Calcium depletion enhances nectin-1 expression and herpes oncolytic therapy of squamous cell carcinoma

Attenuated, replication-competent, oncolytic herpes simplex virus type 1 (HSV-1) are effective at infecting and lysing many human malignancies in preclinical studies. Nectin-1 is a cell-surface receptor for HSV-1 envelope glycoprotein D (gD) that also forms a component of intercellular adherens junctions (AJs). We sought to determine if the disruption of AJs in squamous cell carcinoma (SCC) through calcium depletion could be utilized to increase nectin-1 exposure and enhance HSV therapy. NV1023 is a single copy gamma(1)34.5-deleted, lacZ-expressing, oncolytic HSV-1. Calcium depletion caused cell separation and increased nectin-1 expression for three SCC cell lines growing at confluence. NV1023 viral entry, soluble gD protein binding and NV1023 cytotoxicity were all significantly enhanced for these cell lines at low calcium conditions. The increase in NV1023 entry at low calcium conditions was abrogated by nectin-1 antibody blockade. Murine SCC flank tumors treated with ethylenediaminetetraacetic acid (EDTA) showed increased nectin-1 expression and increased susceptibility to NV1023 infection. Combined NV1023 and EDTA intratumoral injections demonstrated significantly enhanced tumor regression as compared to NV1023 alone. These findings establish, as proof-of-principle, that herpes viral receptor expression may be modulated on cancer cells to enhance oncolytic therapy. This strategy might have future application toward improving therapy with a variety of herpes vectors.     

Sensitivity of squamous cell carcinoma lymph node metastases to herpes oncolytic therapy.

PURPOSE: Cancer metastases may have phenotypic and genetic differences from their primary cancers of origin. Engineered, replication-competent, attenuated viruses based on herpes simplex virus-1 (HSV-1) have shown potent oncolytic effects in treating primary tumors in animal tumor models, but their efficacy in treating lymph node metastases is poorly understood. We compared the efficacy of an attenuated oncolytic HSV-1 (NV1023) in treating a series of murine squamous carcinoma cell lines derived from serial implantation and harvest from metastatic lymph nodes. EXPERIMENTAL DESIGN and RESULTS: The auricles of C3H/HeJ mice were implanted with SCCVII. Cervical nodal metastases were isolated, expanded in vitro, and reimplanted into new mice. A series of cell lines (LN1-LN7) were generated through seven serial passages. Cells from higher LN passages showed consistent trends toward increased migratory and invasive ability, increased cell surface nectin-1 (an HSV-1 receptor) expression, and increased glycoprotein D binding. Exposure to NV1023 showed increased viral entry, replication, and cytotoxicity with higher LN passages. Intratumoral injection of NV1023 in a murine flank tumor model caused significantly greater tumor regression and increased viral infection of LN7 compared with SCCVII. CONCLUSIONS: These results show that lymph node metastases may undergo selection for characteristics, including increased nectin-1 expression, that make them more sensitive targets for herpes oncolytic therapy. These findings support the clinical application of these agents for the treatment of lymph node metastases.     

Prodrug bioactivation and oncolysis of diffuse liver metastases by a herpes simplex virus 1 mutant that expresses the CYP2B1 transgene

BACKGROUND: Herpes simplex virus 1 (HSV-1) infection of cancer cells results in viral replication with cell destruction and liberation of progeny virion that infect adjacent tumor cells. rRp450 is a novel replication-conditional HSV-1 mutant that expresses both the endogenous herpes viral thymidine kinase gene and the rat p450 CYP2B1 transgene; p450 bioactivates such cancer prodrugs as cyclophosphamide. METHODS: Viral cytotoxicity and replication assays were performed in colon carcinoma cells as well as primary human hepatocytes. For in vivo studies, diffuse liver metastases were generated by inoculating MC26 cells into the portal system of BALB/c mice. Mice were treated with control media, rRp450, or rRp450 plus cyclophosphamide. RESULTS: Cytopathic effects induced by rRp450 replication in colon carcinoma cells were equivalent to those induced by wild type HSV-1 in vitro. Assays developed to separate cytotoxicity mediated by viral replication from cytotoxicity mediated by chemotherapy confirmed that HSV-1 thymidine kinase bioactivates ganciclovir and CYP2B1 bioactivates cyclophosphamide in rRp450-infected cells. rRp450 mediated cytotoxicity in the presence of cyclophosphamide was increased by 21% to 30% above that achieved by viral replication alone. Cyclophosphamide bioactivation produced bystander killing of colon carcinoma cells but not hepatocytes. In contrast to these effects of cyclophosphamide, rRp450 mediated cytotoxicity was reduced in the presence of ganciclovir. These findings are explained by further experiments showing that bioactivation of cyclophosphamide only minimally affected HSV-1 replication in colon carcinoma cells, whereas bioactivation of ganciclovir markedly attenuated HSV-1 replication. In vivo studies revealed a substantial decrease in hepatic tumor burden in all rRp450-treated animals compared to controls. The addition of cyclophosphamide augmented rRp450's in vivo anti-neoplastic effect. CONCLUSIONS: The rRp450 mutant HSV-1 is highly oncolytic against colon carcinoma cells both in vitro and in vivo. rRp450 displays preferential replication in colon carcinoma cells compared to normal hepatocytes. Activation of cyclophosphamide by the p450 transgene augmented the anti-neoplastic effects of rRp450 without simultaneously decreasing viral replication. Oncolysis induced by HSV-1 replication combined with cyclophosphamide prodrug activation warrants further investigation as a potential therapy for colon carcinoma liver metastases.     

Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil.

Infection of tumor cells by herpes simplex virus 1 (HSV-1) results in cell destruction and production of progeny virion in a process referred to as viral oncolysis. In this study, an HSV-1 mutant (HSV1yCD) was engineered such that the viral ribonucleotide reductase gene is disrupted by sequences encoding yeast cytosine deaminase, which efficiently metabolizes the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). HSV1yCD-infected cells convert 5-FC to 5-FU, which enhances cytotoxicity without significantly reducing viral replication and oncolysis. Oncolysis by a replicating HSV-1 mutant combined with therapeutic transgene delivery represents a new paradigm; HSV1yCD-infected cells are destroyed by viral replication, and uninfected cells are subjected to bystander killing from both progeny virion and extracellular diffusion of 5-FU. In contrast, HSV1yCD-mediated bioactivation of another prodrug, ganciclovir, impairs viral replication. HSV1yCD administered into the portal venous system replicates preferentially in liver metastases rather than normal liver. The anti-neoplastic activity of HSV1yCD combined with systemic 5-FC administration is greater than that achieved with HSV-1 replication alone. Combination oncolysis and prodrug bioactivation leads to significant prolongation of survival in mice with diffuse liver metastases.     

JNK-deficiency enhanced oncolytic vaccinia virus replication and blocked activation of double-stranded RNA-dependent protein kinase

Vaccinia virus has recently been used as an expression vector for gene delivery and an oncolytic agent for cancer therapy. Although it has been established that interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) and RNase L interfere with viral replication, little else is known about the other host factors that might affect viral replication and virus-mediated host cell killing. In this study, we evaluated the roles of c-Jun NH2-terminal kinase (JNK) in oncolytic vaccinia virus replication and vaccinia virus-mediated host cell killing. We found that JNK knockout mouse embryonic fibroblasts (MEFs) were more susceptible to oncolytic vaccinia virus infection than wild-type MEFs. Moreover, viral replication and the production of infectious viral progeny were up to 100-fold greater in JNK-deficient MEFs than in wild-type MEFs. A similar result was observed for wild-type vaccinia virus. The increased killing of infected cells and the production of viral progeny was also observed in wild-type MEFs that had been treated with JNK inhibitors and in human colon cancer cells that had been transfected with dominant-negative JNK constructs. Moreover, testing on several human lung cancer cell lines and HeLa cells showed an inverse correlation between levels of JNK expression and susceptibility to oncolytic vaccinia virus. Our study also revealed that oncolytic virus infection-mediated PKR activation was blocked or diminished in JNK-deficient MEFs. The adenovirus-mediated ectopic expression of human PKR in JNK-deficient MEFs reduced vaccinia virus replication to the levels observed in wild-type MEFs, indicating that JNK is required for vaccinia virus to efficiently activate PKR. Our results demonstrated that the cellular status of JNK function can dramatically affect oncolytic vaccinia virus replication and vaccinia virus-mediated host cell killing. This finding may enable further improvements in oncolytic virotherapy using vaccinia virus.     

Widespread intratumoral virus distribution with fractionated injection enables local control of large human rhabdomyosarcoma xenografts by oncolytic herpes simplex viruses

Novel methods of local control for sarcomas are needed. We investigated the antitumor effect of two related herpes simplex virus (HSV) mutants, NV1020 and NV1066, on human rhabdomyosracoma cells and xenografts. Cell death correlated with virus replication and apoptosis in cultured cells and tumors. Complete regression was seen in all tumors <250 mm(3) following a single injection, yet only half of tumors >250 mm(3) showed a complete response. Fractionation of the virus dose into five injection sites did not increase transduction efficiency, transgene expression, or virus production, but did yield more widespread intratumoral distribution. Despite the same total dose of virus, improved control of large tumors was seen using fractionated injections as all large tumors (500-700 mm(3)) had durable, complete regression. Our data suggest that oncolytic HSVs may be useful for local control of bulky rhabdomyosarcoma tumors and that fractionated virus administration results in a more widespread virus infection and better tumor control. Therefore, strategies to maximize intratumoral virus distribution at initial delivery should be sought.     

Oncolysis of diffuse hepatocellular carcinoma by intravascular administration of a replication-competent, genetically engineered herpesvirus

Herpes simplex virus type 1 (HSV-1) replication within tumors can mediate tumor regression (oncolysis). The genetically engineered, HSV-1 mutant rRp450 does not express viral ribonucleotide reductase and is therefore replication conditional. During the course of infection, rRp450 expresses the cytochrome P450 transgene and HSV-1 thymidine kinase gene, thereby enabling it to bioactivate the prodrugs cyclophosphamide and ganciclovir, respectively. rRp450 replication in hepatocellular carcinoma (HCC) cells is cytotoxic and liberates progeny virion that infect adjacent tumor cells. rRp450-mediated oncolysis is enhanced in the presence of cyclophosphamide, whereas it is inhibited in the presence of ganciclovir. As a consequence of defective viral ribonucleotide reductase expression, the yield of rRp450 progeny virions from infection of HCC cells is 3 to 4 log orders greater than that from infection of normal hepatocytes. This is associated with dramatic tumor reduction of diffuse HCC after a single intravascular administration of rRp450. rRp450 holds the promise of the dual therapeutic benefit of selective oncolysis and P450 transgene delivery.     

A herpes simplex virus type 1 mutant deleted for gamma34.5 and LAT kills glioma cells in vitro and is inhibited for in vivo reactivation

To create an oncolytic herpes simplex virus type 1 (HSV-1) that is inhibited for reactivation, we constructed a novel herpes recombinant virus with deletions in the gamma34.5 and LAT genes. The LAT gene was replaced by the gene for green fluorescent protein, thereby allowing viral infection to be followed. This virus, designated DM33, is effective in killing primary and established human glioma cell lines in culture. DM33 is considerably less virulent following intracerebral inoculation of HSV-susceptible BALB/c mice than the wild-type HSV-1 strain McKrae. The safety of this virus is further supported by the retention of its sensitivity to ganciclovir and its relatively limited toxicity against cultured human neuronal cells, astrocytes, and endothelial cells. The ability of DM33 to spontaneously reactivate was tested in a rabbit ocular infection model that accurately depicts human herpes infection and reactivation. Following ocular infection of rabbits, spontaneous reactivation was detected in 83% (15/18) of the eyes infected with wild-type McKrae. In contrast, none of the eyes infected with DM33 had detectable reactivation. The efficacy of this virus in cultured human glioma cell lines, its safety, confirmed by its inability to reactivate, and its attenuated neurovirulence make DM33 a promising oncolytic agent for tumor therapy.     

Combined oncolytic and vaccination activities of parvovirus H-1 in a metastatic tumor model

Oncolytic viruses have emerged as a novel class of potent anticancer agents offering an improvement on chemo- and radiotherapy in terms of tumor targeting and reduction of side-effects. Among these agents, autonomous parvoviruses have attracted the attention of researchers for their ability to preferentially replicate in and kill transformed cells, and to suppress tumors in the absence of adverse reactions in various animal models. We have previously shown that lethally irradiated autologous tumor cells can support parvovirus H-1PV production and serve as carriers to deliver progeny H-1PV into the vicinity of lung metastases in a rat tumor model, resulting in H-1PV infection of and multiplication in metastatic cells. It is known that irradiated autologous (neoplastic) cells can also act as a therapeutic vaccine against the original tumor. Yet the ability of these cells to suppress metastases in the above model was found to be much increased as a result of their H-1PV infection. This prompted us to determine whether H-1PV boosted the tumor-suppressing capacity of the autologous vaccine by increasing its immunogenic potential and/or by making it a factory of oncolytic viruses able to reach and destroy the metastases. Both effects could be dissociated in the presence of neutralising antibodies which either prevent the progeny viruses from spreading to metastatic cells, or deplete the CD8 effector cells from the immune system. This strategy revealed that the H-1PV infection of tumor cells enhanced their ability to trigger an immune response for which uninfected tumor cells could be the targets, thereby amplifying and taking over from the direct viral oncolytic activity. This dual oncolytic/vaccinal effect of H-1PV holds out promises of clinical applications to cancer therapy.